Brain targeted low molecular weight hydrophobic anti oxidant compounds

ABSTRACT

Novel brain targeted low molecular weight, hydrophobic antioxidants and use of antioxidants in treatment of central nervous system neurodegenerative disorders such as Parkinson&#39;s, Alzheimer&#39;s and Creutzfeldt-Jakob&#39;s diseases and in treatment of conditions of peripheral tissues, such as acute respiratory distress syndrome, amyotrophic lateral sclerosis, atheroscierotic cardiovascular disease and multiple organ dysfunction, in which oxidants are overproduced.

[0001] This is a Continuation of U.S. patent application Ser. No.09/322,980, filed Jun. 1, 1999, which is a Continuation-In-Part ofPCT/US97/23997, filed Dec. 23, 1997, now expired, which claims priorityfrom U.S. patent application Ser. No. 08/773,153, filed Dec. 26, 1996,now U.S. Pat. No. 5,874,468, issued Feb. 23, 1999.

FIELD AND BACKGROUND OF THE INVENTION

[0002] The present invention relates to composition and use of novelantioxidant compounds, also referred herein as antioxidants. Moreparticularly, the present invention relates to novel brain targeted lowmolecular weight, hydrophobic antioxidants and their use in treatment ofcentral nervous system neurodegenerative disorders such as Parkinson's,Alzheimer's and Creutzfeldt-Jakob's diseases. The compounds according tothe present invention can also be used as antioxidants in treatingconditions of peripheral tissues, such as acute respiratory distresssyndrome, amyotrophic lateral sclerosis, atheroscierotic cardiovasculardisease and multiple organ dysfunction.

[0003] Correlation between oxidative stress and variousneurodegenerative pathologies.

[0004] In the last few years evidences have accumulated which connectoxidative stress (OS) with the pathogenesis of Parkinson's, Alzheimer'sCreutzfeldt-Jakob's diseases and other human neurodegenerative disorders(Olanow, 1990, 1993; Fahn and Cohen, 1992; Cafe et al., 1996, Brown etal., 1996; Thomas et al., 1996).

[0005] These studies were initiated (i) since outo-oxidation of levodopaand dopamine is known to produce oxygen free radicals, H₂O₂, quinonesand semiquinones, the later two are high molecular weight polymerspossessing an aromatic structure and are therefore potentially toxic and(ii) since post-mortem studies in Parkinson's disease patients showed adramatic decline in the levels of endogenous reduced glutathione (GSH),which is, as is further delineated hereinbelow, essential formaintaining the oxidative state of the cells. The decrease in reducedglutathione levels progresses from the pre symptomatic Parkinson'sdisease condition to the advanced clinical Parkinson's diseasecondition.

[0006] Two possible explanations may account for the role played byoxidative stress in the pathogenesis of Parkinson's disease.

[0007] According to a first hypothesis it is assumed that cells of thesubstantia nigra (SN), are constantly under oxidative stress due to theoxidation of the catechol ring of dopamine. Dopamine, like othercatecholamines, undergoes spontaneous oxidation to form semiquinones,oxygen free-radicals and H₂O₂ as metabolic by-products. In addition, oneof the disposal routs for dopamine is its enzymatic oxidation by MAO(monamine oxidase) type A or B which, like the spontaneous oxidation,creates semiquinones, oxygen free radicals and H₂O₂. These products maycause accumulative oxidation damage within the substantia nigra cells,and eventually lead to cell death. Non-affected cells increase theturnover of dopamine, which in turn, generates more toxic free radicals.Indeed, it was shown that in the presence of H₂O₂ and copper ions,dopamine as well as L-dopa (levodopa) cause oxidative damage to DNA(Jenner 1994; Spencer et al., 1994).

[0008] According to another hypothesis it is assumed that Parkinson'sdisease is caused by a substance of an unknown composition, similar tothe toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which isenzymatically converted to the toxic metabolite 1-methyl-4-phenylpyridine (MPP⁺) by MAO type B. Since this reaction releases free oxygenradicals, thereby increasing the oxidative stress imposed on the cells,similar mechanisms might affect the nigral cells in Parkinson's disease.If this type of a process indeed occurs in Parkinsonian brains, thanagain, it would create a high level of oxidative stress in thedopaminergic cells at the substantia nigra.

[0009] The following findings demonstrate a strong indication foroxidative stress as a possible cause for the pathogenesis of Parkinson'sdisease.

[0010] First, the iron content in the substentia Nigra of Parkinson'sdisease patients was found to be significantly higher while ferritincontent, the protein which bind free iron ions within tissues, was foundto be significantly lower then normal values (Olanow 1990; 1993 andJenner 1994). These two phenomena indicate a situation where the amountof free iron which acts as a catalyst in oxidation reactions, isabnormally high and thus may contribute to the speed of oxidationreactions at the substantia nigra of Parkinson's disease patients. Theabove indication was given support when it was shown that injection offree iron directly into the substantia nigra of rodents caused theappearance of Parkinsonian symptoms, which symptoms could be overcome byaddition of transferrin, the protein which binds free iron in the bloodplasma (Jenner 1994).

[0011] Second, one of the protective mechanisms against oxidationprocesses in the brain is reduced glutathione (GSH), which uponoxidation to oxidized glutathione (GS), acts as a reducing agent. In thesubstantia nigra of Parkinson's disease patients the level of reducedglutathione is significantly low, whereas the level of oxidizedglutathione remains normal. Hence the oxidation potential in thesubstantia nigra of Parkinson's disease patients is low. This change inthe level of reduced glutathione is in all likelihood, specific forParkinson's disease. The oxidation products which are formed duringspontaneous and enzymatic oxidation of dopamine, as describedhereinabove, lower the level of reduced glutathione, and therebyincrease the ratio of oxidized/reduced glutathione. Since the level ofγ-glutamyl-cysteine synthetase, the enzyme which is a rate limitingenzyme in the biochemical pathway of glutathione synthesis, is normal,the ratio stays high and induces a state of oxidative stress in thecells. It is interesting that the level of the enzyme which isresponsible for the removal of oxidized glutathione, γ-glutamyltranspeptidase in the cells is higher, as if the cells attempt toovercome the increased oxidative stress by trying to get rid of oxidizedglutathione (Sian et al., 1994).

[0012] Third, additional evidence for the abnormally high oxidativestress in the Parkinsonian brain comes from a study of lipid oxidationproducts in the substantia nigra of Parkinson's disease patients. Ingeneral, the level of unsaturated fatty acids is low in the substantianigra, however, the level of lipid hydroperoxides which are theoxidation products of unsaturated fatty acids is high in the substantianigra of Parkinson's patients. This finding indicates the presence of anabnormally higher frequency of oxidation processes in the Parkinsoniansubstantia nigra (Jenner, 1994).

[0013] And finally, one of the animal models which are currently usedfor the study of Parkinson's disease is created by the injection of6-hydroxy-dopamine. Like dopamine, this false neurotransmitter elevatesthe level of the oxidation products during its degradation, thus leadingto cell death. Since the biological half life of 6-hydroxy-dopamine ismuch longer and since it is readily taken-up by the cells, it increasesthe rate by which the animal develops the symptoms of the disease.

[0014] The different pathological makers of various neurodegenerativediseases e.g., Lewy bodies, plaques, etc., indicate different causalfactors in the initiation of these diseases. However, there is growingevidence that once initiated, the progression of a large number ofneurodegenerative diseases, is quite similar.

[0015] Although the characteristic symptoms are descriptive for eachneurodegenerative disease, it appears that elevation of the oxidativestate of the cells at specific regions in the brain is an importantfactor in the etiology of Parkinson's disease, basal gangliadegenerative diseases, motoneuron diseases, Alzheimer's and also theCreutzfeldt-Jakob's disease.

[0016] An indication for a role played by oxidative stress in thepathogenesis of Alzheimer's disease was found while in a recent study,the relationship between the β-amyloid protein fragments and oxygenradical formation was tested in a system that is highly sensitive andresponds to free oxygen radicals. This system utilizes the vasoactivityof the blood vessel which, in the presence of β-amyloid, enhances thephenylephrine mediated contraction of the vessels. Pre treatment of theblood vessel with superoxide dismutase (SOD), an enzyme that scavengesfree oxygen radicals, eliminated the effect of β-amyloid, namely, therewas no enhancement of vasoconstriction. Whereas, if SOD was added aftertreatment with β-amyloid protein, the protective effect of the radicalscavenger was abolished (Thomas et al., 1996). Recently, other studieshave shown that oxidative stress and free radicals production are linkedto β-amyloid fragment which includes amino acids 25-35 and maycontribute to neurodegenerative events associated with Alzheimer'sdisease (Cafe et al., 1996).

[0017] Possible indications for a role played by oxidative stress in thepathogenesis of Scrapie, spongyform encephalopathy (BSE) andCreutzfeldt-Jakob's diseases are listed hereinbelow.

[0018] In a recent study, it was demonstrated that the toxic effect ofScrapie requires the presence of microglia cells which respond to aprion protein fragment (PrP106-126) by increasing their oxygen radicalproduction. Interestingly, all these effects were absolutely dependenton mice that express the prion protein PrP^(c) (Brown et al., 1996). Thecontribution of progressive oxidative stress to the state of variousdiseases and to mechanism of cell death is further demonstrated in astudy by P. Jenner in The Lancet (1994) 344, 796-798, which isincorporated by reference as if fully set forth herein.

[0019] New therapeutic aspects.

[0020] The use of glial cell-derived neurotrophic factor (GDNF) wasestablished as a potential stimulant for the increase of dopamine levelsin midbrain of rhesus monkeys (Gash et al., 1996). This study whichextends previous results obtained with rodents, is promising as apotential treatment for Parkinson's disease. However, like any otherprotein, GDNF cannot cross the blood brain barrier. Therefore, it cannot be taken orally or be injected systemically. The only possible modeof administration would thus be via an intracerebral injection whichwould constitute a main drawback for such a treatment.

[0021] Similarly, in other neurodegenerative diseases such asAlzheimer's and Creutzfeldt-Jakob's, where the theory of free oxygenradicals appears to play a major role, there is no major breakthrough intherapy.

[0022] To overcome high oxidative stress it would be beneficial toaugment the reduced state of the cells at the central nervous system(CNS). One of the possible ways to do it is by increasing the level ofreduced glutathione or other scavengers of free radicals and free oxygenin the brain.

[0023] In general, in order to lower oxidative stress levels, variousantioxidants are being used. The most common are vitamin E and vitaminC. However, vitamin E was found to be ineffective at decreasing theoxidative stress at the substantia nigra (The Parkinson Study Group,1993, Offen et al., 1996) since this compound, although capable ofcrossing the blood brain barrier, is trapped in the cell membrane andtherefore does not reach the cytoplasm where its antioxidant propertiesare needed. Vitamin C does not cross the blood brain barrier andtherefore, cannot be used effectively for neurodegenerative diseases ofcentral origin.

[0024] Recently, a similar approach for reducing the levels of freeoxygen, was taken for the treatment of asthma (Bundy et al., 1995). Areactive oxygen inhibitor was synthesized(2,4-diaminopyrrolo-[2,3-d]pyrimidines) and after a successfulpharmacological bio-availability and toxicity tests was selected forclinical evaluation.

[0025] A somewhat different approach involves stimulating the productionof endogenous antioxidants, especially reduced glutathione. To this enda drug known as Procysteine which boosts cellular production ofglutathione by loading the cells with cysteine is under clinical trialsthese days by Free Radical Sciences Inc. (CA, US) to treat conditions ofacute respiratory distress syndrome (ARDS) which includes overproductionof oxidants or reactive oxygen species by the immune system. Otherconditions in which overproduction of oxidants is experienced includebut are not limited to amyotrophic lateral sclerosis, atheroscieroticcardiovascular disease and multiple organ dysfunction. See, for example,Charles Craig, 1996.

[0026] There is thus a widely recognized need for, and it would behighly advantageous to have novel antioxidant compounds and methods foruse of antioxidants in treatment of central nervous systemneurodegenerative disorders such as Parkinson's, Alzheimer's andCreutzfeldt-Jakob's diseases and in treating conditions of peripheraltissues, such as acute respiratory distress syndrome, amyotrophiclateral sclerosis, atheroscierotic cardiovascular disease and multipleorgan dysfunction, which compounds act as oxygen scavengers to lower theoxidative stress within and in the vicinity of affected cells andeventually to stop cell death which is associated with oxidative stressin the brain and/or peripheral tissues.

SUMMARY OF THE INVENTION

[0027] According to the present invention there are provided brainand/or peripheral tissues targeted, low molecular weight, hydrophobicantioxidant compounds. Further provided are methods of using same fortreatment of various etiologies. Particularly, these etiologies areneurodegenerative disorders, in which disorders pathology in the brainis associated with oxidative stress.

[0028] According to further features in preferred embodiments of theinvention described below provided is a compound selected from the groupconsisting of

[0029] According to further features in preferred embodiments of theinvention described below provided is a method of preparing N-acetylcysteine ethyl ester, which is the first compound above, the methodcomprising the steps of (a) mixing N-acetyl cysteine with a cooledsolution of thionyl chloride and absolute ethanol; (b) refluxing themixture; and (c) removing volatiles from the mixture for obtaining afirst residue.

[0030] According to still further features in the described preferredembodiments the method further comprising the steps of (d) dissolvingthe first residue in water; and (e) extracting the first residue fromthe water with methylene chloride.

[0031] According to still further features in the described preferredembodiments the method further comprising the step of (f) drying theextract for obtaining a second residue.

[0032] According to still further features in the described preferredembodiments the method further comprising the step of (g) crystallizingthe second residue from petroleum ether.

[0033] According to further features in preferred embodiments of theinvention described below provided is a method of preparing N-acetylβ,β-dimethyl cysteine ethyl ester, which is the second compound above,the method comprising the steps of (a) mixing N-acetyl β,β-dimethylcysteine with a cooled solution of thionyl chloride and absoluteethanol; (b) refluxing the mixture; and (c) removing volatiles from themixture for obtaining a first residue.

[0034] According to still further features in the described preferredembodiments the method further comprising the steps of (d) dissolvingthe first residue in water; and (e) extracting the first residue fromthe water with methylene chloride.

[0035] According to still further features in the described preferredembodiments the method further comprising the step of (f) drying theextract for obtaining a second residue.

[0036] According to still further features in the described preferredembodiments the method further comprising the step of (g) crystallizingthe second residue from a methanol water solution.

[0037] According to further features in preferred embodiments of theinvention described below provided is a method of preparing N-acetylglutathione amide, which is the ninth compound above, the methodcomprising the steps of (a) bobbling ammonia gas through cooled ethanol;(b) adding N-acetyl glutathione ethyl ester, which is the seventhcompound above; (c) sealing a container holding the reaction; and (d)removing access ammonia and ethanol.

[0038] According to further features in preferred embodiments of theinvention described below provided is a method of preparing N-acetylcysteine amide, which is the tenth compound above, the method comprisingthe steps of (a) bobbling ammonia gas through cooled ethanol; (b) addingN-acetyl cysteine ethyl ester, which is the first compound above; (c)sealing a container holding the reaction; and (d) removing accessammonia and ethanol.

[0039] According to further features in preferred embodiments of theinvention described below provided is a method of preparing N-acetyl β,βdimethyl cysteine amide, which is the eleventh compound above, themethod comprising the steps of (a) bobbling ammonia gas through cooledethanol; (b) adding N-acetyl β,β dimethyl cysteine ethyl ester, which isthe second compound above; (c) sealing a container holding the reaction;and (d) removing access ammonia and ethanol.

[0040] According to further features in preferred embodiments of theinvention described below provided is a method of reducing oxidativestress in the brain of an organism having a blood brain barrier, themethod comprising the step of administering a compound to the organism,the compound having (a) a combination of molecular weight and membranemiscibility properties for permitting the compound to cross the bloodbrain barrier of the organism; (b) a readily oxidizable chemical groupfor exerting antioxidation properties; and (c) a chemical make-up forpermitting the compound or its intracellular derivative to accumulatewithin the cytoplasm of cells.

[0041] According to still further features in the described preferredembodiments the readily oxidizable chemical group is a sulfhydril group.

[0042] According to still further features in the described preferredembodiments the chemical make-up is selected having an ester moietywhich is removable by hydrolysis imposed by intracellular esterases.

[0043] According to still further features in the described preferredembodiments the administration is systemic (i.e., not directly to thecentral nervous system, e.g., orally or by intravenous injection).

[0044] According to still further features in the described preferredembodiments the ester moiety is selected from the group consisting ofalkyl ester and aryl ester.

[0045] According to still further features in the described preferredembodiments the alkyl and aryl esters are selected from the groupconsisting of methyl ester, ethyl ester, hydroxyethyl ester, t-butylester, cholesteryl ester, isopropyl ester and glyceryl ester.

[0046] According to still further features in the described preferredembodiments the organism is a human being.

[0047] According to still further features in the described preferredembodiments the oxidative stress in the brain is a pathology caused by aneurodegenerative disorder.

[0048] According to still further features in the described preferredembodiments the neurodegenerative disorder is selected from the groupconsisting of Parkinson's disease, Alzheimer's disease, basal gangliadegenerative diseases, motoneuron diseases, Scrapie, spongyformencephalopathy and Creutzfeldt-Jakob's disease.

[0049] According to further features in preferred embodiments of theinvention described below provided is a method of reducing oxidativestress in a tissues of an organism, the method comprising the step ofadministering a compound to the organism, the compound is selected fromthe group consisting of

[0050] According to further features in preferred embodiments of theinvention described below provided is a method of reducing orabolishing, extracellular or intracellular, natural or induced oxidationeffects imposed on cells, the method comprising the step of subjectingthe cells to a compound selected from the group consisting of N-acetylcysteine ethyl ester, β,β-dimethyl cysteine ethyl ester,N-acetyl-β,β-dimethyl cysteine, glutathione ethyl ester, N-acetylglutathione ethyl ester, N-acetyl glutathione, N-acetyl (o ethyl ester)glutathione, N-acetyl (o ethyl ester) glutathione ethyl ester, N-acetylglutathione amide, N-acetyl cysteine amide, N-acetyl β,β dimethylcysteine amide and N-acetyl cysteine glycine amide.

[0051] According to still further features in the described preferredembodiments the cells are of an organism suffering from a conditionassociated with over production of oxidants.

[0052] According to still further features in the described preferredembodiments the organism is a human being and the condition is selectedfrom the group consisting of acute respiratory distress syndrome,amyotrophic lateral sclerosis, atherosclerotic cardiovascular disease,multiple organ dysfunction, Parkinson's disease, Alzheimer's disease,basal ganglia degenerative diseases, motoneuron diseases, Scrapie,spongyform encephalopathy and Creutzfeldt-Jakob's disease.

[0053] According to further features in preferred embodiments of theinvention described below provided is a method of therapeutically orprophylactically treating an individual known to have a central nervoussystem neurodegenerative disorder associated with oxidative stress, themethod comprising the step of administering a pharmaceutical compositionto the individual, the pharmacological composition including a compound,the compound having (a) a combination of molecular weight and membranemiscibility properties for permitting the compound to cross the bloodbrain barrier of the individual; (b) a readily oxidizable chemical groupfor exerting antioxidation properties; and (c) a chemical make-up forpermitting the compound or its intracellular derivative to accumulatewithin brain cells of the individual.

[0054] According to still further features in the described preferredembodiments the central nervous system neurodegenerative disorderassociated with oxidative stress is selected from the group consistingof Parkinson's disease, Alzheimer's disease, basal ganglia degenerativediseases, motoneuron diseases, Scrapie, spongyform encephalopathy andCreutzfeldt-Jakob's disease.

[0055] According to still further features in the described preferredembodiments the pharmacological composition further includes apharmacological carrier.

[0056] According to still further features in the described preferredembodiments the pharmacological carrier is selected from the groupconsisting of a thickener, a carrier, a buffer, a diluent, a surfaceactive agent and a preservatives.

[0057] According to still further features in the described preferredembodiments the administration is systemic.

[0058] According to still further features in the described preferredembodiments the systemic administration is selected from the groupconsisting of topical administration, oral administration,administration by inhalation, and parenteral administration.

[0059] According to further features in preferred embodiments of theinvention described below provided is a pharmaceutical composition forreducing or abolishing, extracellular or intracellular, natural orinduced oxidation effects imposed on cells of an individual, thecomposition comprising a compound selected from the group consisting ofN-acetyl cysteine ethyl ester, β,β-dimethyl cysteine ethyl ester,N-acetyl-β,β-dimethyl cysteine, glutathione ethyl ester, N-acetylglutathione ethyl ester, N-acetyl glutathione, N-acetyl (o ethyl ester)glutathione, N-acetyl (o ethyl ester) glutathione ethyl ester, N-acetylglutathione amide, N-acetyl cysteine amide, N-acetyl β,β dimethylcysteine amide and N-acetyl cysteine glycine amide.

[0060] According to further features in preferred embodiments of theinvention described below provided is a compound for therapeutically orprophylactically treating an individual known to have a central nervoussystem neurodegenerative disorder associated with oxidative stress, thecompound comprising (a) a combination of molecular weight and membranemiscibility properties for permitting the compound to cross the bloodbrain barrier of the individual; (b) a readily oxidizable chemical groupfor exerting antioxidation properties; and (c) a chemical make-up forpermitting the compound or its intracellular derivative to accumulatewithin brain cells of the individual.

[0061] According to still further features in the described preferredembodiments the cells are of an organism suffering from a conditionassociated with over production of oxidants.

[0062] According to still further features in the described preferredembodiments the organism is a human being and the condition is selectedfrom the group consisting of acute respiratory distress syndrome,amyotrophic lateral sclerosis, atheroscierotic cardiovascular disease,multiple organ dysfunction, Parkinson's disease, Alzheimer's disease,basal ganglia degenerative diseases, motoneuron diseases, Scrapie,spongyform encephalopathy and Creutzfeldt-Jakob's disease.

[0063] According to further features in preferred embodiments of theinvention described below provided is a pharmaceutical composition fortherapeutically or prophylactically treating an individual known to havea central nervous system neurodegenerative disorder associated withoxidative stress, the composition comprising a compound, the compoundhaving (a) a combination of molecular weight and membrane miscibilityproperties for permitting the compound to cross the blood brain barrierof the individual; (b) a readily oxidizable chemical group for exertingantioxidation properties; and (c) a chemical make-up for permitting thecompound or its intracellular derivative to accumulate within braincells of the individual.

[0064] The present invention successfully addresses the shortcomings ofthe presently known configurations by providing compounds,pharmaceutical compositions containing the compounds, methods ofpreparing the compounds and of using the compounds for therapeutic andprophylactic treatments of oxidative stress associated central nervoussystem neurodegenerative disorders and other etiologies, which atpresent have no known adequate treatment.

[0065] It is one object of the invention to provide novel antioxidantcompounds.

[0066] It is another object of the invention to provide novel bloodbrain barrier crossing antioxidant compounds.

[0067] It is still another object of the invention to providepharmaceutical compositions containing the novel compounds.

[0068] It is a further object of the invention to provide methods ofusing antioxidant compounds capable of crossing the blood brain barrierfor therapeutic or prophylactic treatment of central nervous systemneurodegenerative disorders.

[0069] It is still a further object of the invention to provide a methodfor treating cells subjected to extracellular or intracellular oxidativestress in both central nervous system neurodegenerative disorders and indisorders of peripheral tissues.

[0070] These and other objects of the invention are further delineatedhereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0071] The invention herein described, by way of example only, withreference to the accompanying drawings, wherein:

[0072]FIG. 1 presents [³H]-thymidine uptake by PC12 cells treated invitro with 0.5 mM dopamine which confers extracellular oxidative stressby forming oxidation products during its oxidation in the medium rescuedwith various concentrations of compounds A-D;

[0073]FIG. 2 presents [³H]-thymidine uptake by PC12 cells treated invitro with 0.5 mM 6-hydroxy-dopamine, which confers intracellularoxidative stress by first entering the cytoplasm and then formingoxidation products during its oxidation in the cytoplasm, protected withvarious concentrations of compounds A-D and exogenous reducedglutathione (GSH); and

[0074]FIG. 3 presents the ratio of endogenous reduced glutathione levelsin striatum/serum in two mice injected with 100 mg/kg of compound A in3% DMSO, 100 mg/kg of reduced glutathione in 3% DMSO, and 3% DMSOinjected as a control group, wherein the ratio obtained is marked at thetop of the columns. The results represent two animals where eachstriatum taken separately.

[0075]FIG. 4 demonstrates that Compund J at as low as 0.1 mM protect NBcells against the toxicity (>50%) of DA, L-dopa (levodopa), 6-OHDA(0.1-0.25 mM) and MPP⁺ (0.5-2 mM). Cell survival was monitored by theneutral red assays.

[0076]FIG. 5a shows HPLC profile of purified compund J.

[0077]FIG. 5b shows HPLC profile of a brain extract of a mouse 15minutes following IP injection of compound J.

[0078]FIG. 6 shows the concentration of compound J in brain extracts ofmice 15 minutes following IP injection of compound J at the amountsindicated.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0079] The present invention is of composition and use of antioxidantcompounds. Specifically, the compounds of the present invention are lowmolecular weight, hydrophobic antioxidants which can be used fortreatment of various etiologies. More specifically, the compounds of thepresent invention can be used for treatment of neurodegenerativedisorders, in which disorders pathology in the brain is associated withoxidative stress and for treatment of peripheral tissues in conditionsassociated with overproduction of oxidants.

[0080] Thus, the compounds according to the present invention can beused to treat central nervous system neurodegenerative disorders suchas, but not limited to, Parkinson's, Alzheimer's and Creutzfeldt-Jakob'sdiseases, and peripheral tissue disorders such as, but not limited to,acute respiratory distress syndrome, amyotrophic lateral sclerosis,atheroscierotic cardiovascular disease and multiple organ dysfunction,all of which were previously shown to be associated with formationand/or overproduction of oxidants.

[0081] The principles of operation of the compounds according to thepresent invention may be better understood with reference to thedrawings and accompanying descriptions.

[0082] Antioxidant compounds are used according to the present inventionto relieve oxidation stress within cells. Prefered compounds which areused to relieve oxidation stress according to the present invention (i)has a combination of molecular weight and membrane miscibilityproperties rendering it capable of crossing the blood brain barrier;(ii) includes a readily oxidizable (i.e., reduced) chemical group, suchas, but not limited to, a sulfhydryl (—SH) group, for exerting itsantioxidation properties and (iii) has a chemical make-up for permittingit or its cellular derivative(s) to accumulate within the cytoplasm ofcells, such as brain cells. Collectively, these properties render thecompounds suitable for treatment of neurodegenerative disorder of thecentral nervous system.

[0083] Yet, these compounds are also suitable for treating conditions inwhich peripheral tissues, such as, but not limited to, the lungs and/orheart, are damaged due to overproduction of oxidants (i.e., reactiveoxygen species), which is the case in, for example, acute respiratorydistress syndrome, amyotrophic lateral sclerosis, atheroscieroticcardiovascular disease and multiple organ dysfunction.

[0084] Compounds which have the above listed properties are for example:

[0085] (i) N-acetyl cysteine ethyl ester—C₇H₁₁NO₃S— of a formula(compound A):

[0086] (ii) β,β-dimethyl cysteine ethyl ester or N-acetyl-penicillamineethyl ester—C₉H₁₈NO₃S— of a formula (compound B):

[0087] (iii) N-acetyl-β,β-dimethyl cysteine orN-acetyl-penicillamine—C₇H₁₃NO₃S— of a formula (compound C):

[0088] (iv) Glutathione ethyl ester—C₁₂H₂₁N₃O₆S— of a formula (compoundD):

[0089] (v) N-acetyl glutathione ethyl ester—C₁₄H₂₃N₃O₇S— of a formula(compound E):

[0090] (vi) N-acetyl glutathione—C₁₂H₁₉N₃O₇S— of a formula (compound F):

[0091] (vii) N-acetyl o-glutamyl ethyl ester cysteinyl glycyl ethylester or N-acetyl (o-ethyl ester) glutathione ethyl ester—C₁₆H₂₇N₃O₇S—of a formula (compound G):

[0092] (viii) N-acetyl o-glutamyl ethyl ester cysteinyl glycyl orN-acetyl (o-ethyl ester) glutathione—C₁₄H₂₃N₃O₇S— of a formula (compoundH):

[0093] Additional compounds which may serve as antioxidants according tothe present invention are:

[0094] (ix) N-acetyl glutathione amide—C₁₂H₂₁N₅O₅S— of a formula(compound I):

[0095] (x) N-acetyl cysteine amide—C₅H₁₀N₂O₂S— of a formula (compoundJ):

[0096] (xi) N-acetyl β,β dimethyl cysteine amide—C₇H₁₅N₂O₂S— of aformula (compound K):

[0097] (xii) N-acetyl cysteine glycine amide—C₇H₁₂N₃O₃S— of a formula(compound L):

[0098] These compounds are used according to the invention asantioxidants which cross the blood brain barrier, for relievingoxidative stress, a common propagator of many neurodegenerativediseases. However, it will be appreciated by one ordinarily skilled inthe art, that compounds having the above listed properties may also beused as antioxidants in other etiologies caused by oxidation processesin for example peripheral tissues, i.e., tissues other than centralnervous system tissues.

[0099] According to a preferred embodiment of the invention, thecompound is a pro-drug which penetrates the cells due to its solubilityin the cell membrane and is hydrolyzed once inside the cell, exerting adrug having the antioxidant activity. For example compounds A, B, D, E,G and H above are pro-drug compounds.

[0100] Compounds A, B, E, G and H above are novel pro-drug compounds,and their hydrolytic products ethanol and N-acetyl-cysteine (forcompound A); ethanol and N-acetyl-penicillamine (for compound B);ethanol and N-acetyl glutathione (for compounds E, G and H) are knownnot to be toxic. The lethal dose 50% (LD50) value for N-acetyl-cysteineis 5,050 mg/kg. N-acetyl-penicillamine is available as an oralmedication distributed under the generic name cuprimine by variousmanufacturers. Whereas N-acetyl glutathione and ethanol are both wellknown to be non-toxic substances.

[0101] A pro-drug according to the invention includes at least one estermoiety such as an alkyl ester or an aryl ester, e.g., methyl ester,ethyl ester, hydroxyethyl ester, t-butyl ester, cholesteryl ester,isopropyl ester and glyceryl ester. Preferably the pro-drug includes anethyl ester moiety which, on one hand, neutralizes the charge of thecarboxylic group(s) and on the other hand, when hydrolyzed within thecells release ethanol which is a substance known not to be toxic to thecells.

[0102] Upon entering the cytoplasm of a cell, the pro-drug isde-esterified by one or various intracellular esterases, to release thedrug which has at least one carboxyl moiety (—COOH) and a by-product(typically ethanol) which contains the hydroxyl moiety (—OH). Thecarboxylic group(s) of the drug is typically negatively charged and thedrug therefore is trapped within the cell, where it is to exert itsantioxidative properties.

[0103] Further according to the invention there is provided a method forpreparing compounds A and B. The method includes the following steps.First, N-acetyl cysteine (for compound A) or N-acetyl β,β-dimethylcysteine (for compound B) is mixed with a cooled solution of thionylchloride and absolute ethanol. Second, the mixture is refluxed. Andthird, the volatiles are removed from the mixture for obtaining a firstresidue. Preferably the method further includes the following step.Fourth, the first residue is dissolved in water. And fifth, the firstresidue is extracted from the water with methylene chloride. Preferablythe method further includes the following step. Sixth, the extract isdried to obtain a second residue. Preferably the method further includesthe following step. Seventh, the second residue is crystallized frompetroleum ether (for compound A) or from a methanol water solution (forcompound B). Further detail concerning the method of preparing compoundsA and B are delineated hereinbelow in the Examples section.

[0104] Compound C is described in Biochem. Prep. 3, 111 (1953) and inU.S. Pat. Nos. 2,477,148 and 2,496,426, both are incorporated byreference as if fully set forth herein, and was prepared essentially astherein described. As mentioned above, compound C,N-acetyl-penicillamine, is available as an oral medication distributedunder the generic name cuprimine by various manufacturers.

[0105] Compound D above is commercially available from SigmaBiochemicals, Cat. No. G1404. Compounds D is a pro-drug compound, andits hydrolytic products ethanol and glutathione are well known not to betoxic.

[0106] Compounds E, G and H above are novel glutathione derivatives andcan be prepared, for example, from commercially available building unitsfor Boc and Fmoc chemistry peptide synthesis, as well known in the art.

[0107] Compound F is a glutathione derivative and is described in Levyet al., 1993.

[0108] Further according to the invention there is provided a method forpreparing compounds I, J and K. The method includes the following steps.First, ammonia gas is bubbled through absolute cooled dry ethanol.Second, N-acetyl glutathione ethyl ester (compound G, for synthesis ofcompound I), N-acetyl cysteine ethyl ester (compound A, for synthesis ofcompound J) or N-acetyl β,β dimethyl cysteine ethyl ester (compound B,for synthesis of compound K) is added to the ethanol solution. Third, acontainer holding the reaction is sealed. Fourth, access ammonia andethanol are evaporated and finally the resulting product is lyophilized.Further detail concerning the method of preparing compounds I, J and Kare delineated hereinbelow in the Examples section.

[0109] Further according to the invention there is provided a method forpreparing compound L which is further detailed in the Examples sectionthat follows.

[0110] Any of the glutathione derived compounds (D-H and I ) accordingto the invention may be prepared employing Boc and Fmoc chemistry forpeptide synthesis. This, in turn, permits the inclusion of native Levo(L isomer) and/or non-native Dextro (D isomer) glutamic acid and/orcysteine derivatives or residues within any of these compounds. It willbe appreciated that by replacing the native L configuration by thenon-native D configuration, a compound becomes less recognizable by manyenzymes and its biological half life within the body thereforeincreases. Compounds A-C and J-K also include chiral carbons. Any ofthese carbons may also acquire a D or an L isomreric configuration.

[0111] Thus, compounds A-L above were given chemical names according toall L isomer configurations, i.e., all of their chiral carbon atoms areL isomers. However, as used herein in the specification and in theclaims, these chemical names also refer to any of their D isomer(s)containing chiral atoms.

[0112] As mentioned above, compounds D-H are glutathione derivatives.These compounds and similar glutathione derivative compounds arerepresented by the general formula:

[0113] R1 is selected from the group consisting of a hydrogen atom andan alkyl (e.g., C₁-C₂₀) or aryl (e.g., C₆-C₉) group. Preferably R1 is anethyl group.

[0114] R2 is selected from the group consisting of a hydrogen atom andan alkyl (e.g., C₁-C₂₀) or aryl (e.g., C₆-C₉) group. Preferably R2 is aethyl group.

[0115] Whereas, R3 is selected from the group consisting of a hydrogenatom and an R4-CO (acyl) group, wherein R4 is an alkyl (e.g., C₁-C₂₀) oraryl (e.g., C₆-C₉) group. Preferably R4 is a methyl group. However, anyone of R1, R2 and R4 can independently be a methyl, ethyl, hydroxyethyl,t-butyl, cholesteryl, isopropyl or glyceryl group.

[0116] Compounds A, B and E-L are novel compounds. These compounds arenot listed in the Chemical Abstract.

[0117] For therapeutic or prophylactic treatment of diseases, disordersor medical conditions, the antioxidant compounds of the presentinvention can be formulated in a pharmaceutical composition, which mayinclude thickeners, carriers, buffers, diluents, surface active agents,preservatives, and the like, all as well known in the art.Pharmaceutical compositions may also include one or more activeingredients such as but not limited to antiinflammatory agents,antimicrobial agents, anesthetics and the like in addition to theantioxidant compounds.

[0118] The pharmaceutical composition may be administered in either oneor more of ways depending on whether local or systemic treatment is ofchoice, and on the area to be treated. Administration may be donetopically (including ophtalmically, vaginally, rectally, intranasally),orally, by inhalation, or parenterally, for example by intravenous dripor intraperitoneal, subcutaneous, intramuscular or intravenousinjection.

[0119] Formulations for topical administration may include but are notlimited to lotions, ointments, gels, creams, suppositories, drops,liquids, sprays and powders. Conventional pharmaceutical carriers,aqueous, powder or oily bases, thickeners and the like may be necessaryor desirable.

[0120] Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, sachets,capsules or tablets. Thickeners, diluents, flavorings, dispersing aids,emulsifiers or binders may be desirable.

[0121] Formulations for parenteral administration may include, but arenot limited to, sterile solutions which may also contain buffers,diluents and other suitable additives.

[0122] Dosing is dependent on severity and responsiveness of thecondition to be treated, but will normally be one or more doses per day,with course of treatment lasting from several days to several months oruntil a cure is effected or a diminution of disease state is achieved.Persons ordinarily skilled in the art can easily determine optimumdosages, dosing methodologies and repetition rates.

[0123] Reference in now made to the following examples, which togetherwith the above descriptions, illustrate the invention.

EXAMPLE 1

[0124] Synthesis of N-acetyl Cysteine Ethyl Ester (Compound A)

[0125] N-acetyl cysteine (4.6 mmol) was added in portions to a cooled(e.g., 2-8° C.) solution of 2 ml thionyl chloride and 10 ml absoluteethanol. The resulting mixture was refluxed at 40° C. for 1 hour andthen the volatiles were removed in vacuo. The residue was dissolved in10 ml of water and was extracted twice with 20 ml of methylene chloride.The extract was dried under vacuo. The title compound was crystallizedfrom petroleum ether (fraction 40-60° ) in 55% yield.

[0126] The resulting product has the following characteristics:

[0127] (a) Melting point of 90° C.

[0128] (b) Anal. calculated for C₇H₁₁NO₃S:

[0129] Calculated: C, 43.9 H, 6.8

[0130] Found: C, 42.5 H, 6.0

[0131] (c) Thin layer chromatography in n-butanol/acetic acid/water(4/1/4) was carried out and the Rf value was Rf=0.91. The Rf value ofthe reactant, N-acetyl cysteine is 0.78.

[0132] (d) Nuclear Magnetic Resonance (NMR) in deutaratedtrichloromethane (CDCl3):

[0133] 6.51, 0.7H

[0134] 4.85, 1H, m

[0135] 4.23, 2H, q, J=7.0

[0136] 3.44, 0.4H, d, J=4.4

[0137] 3.22, 2H, t, J=4.4

[0138] 2.06, 3H, S

[0139] 1.30, 3H, t, J=7.0

EXAMPLE 2 Synthesis of N-acetyl β,β-dimethyl Cysteine Ethyl Ester orN-acetyl-penicillamine Ethyl Ester (Compound B)

[0140] N-acetyl β,β-dimethyl cysteine (2.6 mmol) was added in portionsto a cooled (2-8° C.) solution of 2 ml thionyl chloride and 10 mlabsolute ethanol. The resulting mixture was refluxed at 40°0 C. for 1hour and then the volatiles were removed in vacuo. The residue wasdissolved in 10 ml of water and was extracted twice with 20 ml ofmethylene chloride. The extract was dried under vacuo. The titlecompound was crystallized from a methanol-water solution (1/100,fraction 40-60°) in 25% yield.

[0141] The resulting product has the following characteristics:

[0142] (a) Melting point of 180° C.

[0143] (b) Thin layer chromatography in n-butanol/acetic acid/water(4/1/4) was carried out and the Rf value was Rf=0.66. The Rf value ofthe reactant, N-acetyl β,β-dimethyl cysteine is 0.88.

[0144] (c) Nuclear Magnetic Resonance (NMR) in deutarated acetone (D₆)

[0145] 4.79, 1H, d, J=6.0

[0146] 4.17, 2H, q, J=7.0

[0147] 2.81,1H, d, J=6.0

[0148] 1.98, 3H, S

[0149] 1.44, 6H, S

[0150] 1.27, 3H

EXAMPLE 3 Synthesis of N-acetyl Glutathione Amide (Compound I)

[0151] Ammonia gas was bubbled through absolute dry ethanol at −70° C.(dry ice with acetone), for 10 minutes. N-acetyl glutathione ethyl ester(compound G), 350 mg (1 mmol) was added to the cooled ethanol/ammoniasolution and ammonia was continued to bubble through the solution foradditional 10 minutes. Then, the solution was corked and was left atroom temperature. After 16 hours, the flask was opened and access ofammonia and the ethanol were evaporated under reduced pressure. Theproduct was lyophilized. The yield was 84%.

[0152] The resulting product has the following characteristics:

[0153] (a) Thin layer chromatography in n-butanol/acetic acid/water(4/1/4) was carried out and the Rf value was Rf=0.71.

EXAMPLE 4 Synthesis of N-acetyl Cysteine Amide (Compound J)

[0154] Ammonia gas was bubbled through absolute dry ethanol at −70° C.(dry ice with acetone), for 10 minutes. N-acetyl cysteine ethyl ester(compound A), 163 mg (1 mmol) was added to the cooled ethanol/ammoniasolution and ammonia was continued to bubble through the solution foradditional 10 minutes. Then, the solution was corked and was left atroom temperature. After 16 hours, the flask was opened and access ofammonia and the ethanol were evaporated under reduced pressure. Theproduct was lyophilized. The yield was 98%.

[0155] The resulting product has the following characteristics:

[0156] (a) Thin layer chromatography in n-butanol/acetic acid/water(4/1/4) was carried out and the Rf value was Rf=0.70. The Rf value ofthe reactant, N-acetyl cysteine ethyl ester is 0.91.

[0157] Alternatively, a solution of 20% piperidine (4 ml) in 16 ml DMFwas added to Fmoc Rink amide AM resin (2 gram; 1.1 mmole amide) and thereaction was allowed to proceed for 30 minutes. Ac-S-trityl cysteine(1.3 gram, 3.3 mmole) was added with TBTU (1.06 gram) followed bydiisopropyl ethyl amine (1.12 ml). The reaction was carried out for 2hours. The resin was washed with methylene chloride (×6), and then amixture of 1 ml silan/0.5 ml water/19 ml of TFA was added. After 1 hourthe resin was filtered washed with TFA and solvents evaporated. Theproduct was dissolved in water and extracted with methylene chloride.The aqueous solution was thereafter lyophyllized.

[0158] The resulting product has the following characteristics:

[0159] (a) Nuclear Magnetic Resonance (NMR)

[0160] 4.45 t,1,j=6.96 Hz

[0161] 2.81 ABX system, J_(AB)=12.69, J_(AX)+J_(BX)=12.45 Hz

[0162] 2.00 s 3 Hz

EXAMPLE 5 Synthesis of N-acetyl β,βdimethyl Cysteine Amide (Compound K)

[0163] Ammonia gas was bubbled through absolute dry ethanol at −70° C.(dry ice with acetone), for 10 minutes. N-acetyl β,βdimethyl cysteineethyl ester (compound B), 194 mg (1 mmol) was added to the cooledethanol/ammonia solution and ammonia was continued to bubble through thesolution for additional 10 minutes. Then, the solution was corked andwas left at room temperature. After 16 hours, the flask was opened andaccess of ammonia and the ethanol were evaporated under reducedpressure. The product was lyophilized. The yield was 90%.

[0164] The resulting product has the following characteristics:

[0165] (a) Thin layer chromatography in n-butanol/acetic acid/water(4/1/4) was carried out and the Rf value was Rf−0.50. The Rf value ofthe reactant, N-acetyl β,β dimethyl cysteine ethyl ester is 0.66.

EXAMPLE 6 Synthesis of N-acetyl Cysteine Glycine Amide (Compound L)

[0166] A solution of 20% piperidine (4 ml) in 16 ml DMF was added toFmoc Rink glycine amide AM resin (1.1 mmole amide) and the reaction wasallowed to proceed for 30 minutes. Ac-S-trityl cysteine (1.3 gram, 3.3mmole) was then added with TBTU (1.06 gram) followed by diisopropylethyl amine (1.12 ml). The reaction was carried out for 2 hours. Theresin was washed with methylene chloride (×6), and then a mixture of 1ml silan/0.5 ml water/19 ml TFA was added. After 1 hour the resin wasfiltered washed with TFA and solvents were evaporated. The product wasdissolved in water and extracted with methylene chloride. The aqueoussolution was lyophilized.

EXAMPLE 7 In vitro Extracellular Antioxidation by Compounds A-D

[0167] Compounds A-D were assayed in vitro for their extracellularantioxidant activities. The assays were carried out with PC12 cells(Offen et al., 1996) subjected to a high dose of dopamine which confersoxidative stress to these cells by forming oxidation products during itsoxidation in the growth medium, i.e., extracellularly.

[0168] With reference now to FIG. 1. To this end, PC12 cells weresubjected to high concentration of dopamine (0.5 mM) for 24 hours in thepresence of increasing concentrations (0 mM, 0.03 mM, 0.1 mM, 0.3 mM and0.9 mM) of the various compounds A-D. [³H]-thymidine was added to thecells (1 μCi/100,000 cells) six hours before the end of the 24 hoursperiod. Due to the high lipophylicity of compounds A-D, the compoundswere first dissolved in dimethyl sulfoxide (DMSO) and then in water andwere applied to the cells in a final concentration of 3% DMSO. Theeffect of 3% DMSO on the cells was tested separately and the valuespresented in FIG. 1 are after the appropriate corrections.

[0169] [³H]-thymidine uptake was measured in triplicate wells containingcells pretreated with dopamine alone and dopamine with each of compoundsA-D at the concentrations as indicated above. The results presented inFIG. 1 show the mean of triplicate wells taken from three independentcell batches, wherein control represent cells treated only with 3% DMSOand is defined as 100% [³H]-thymidine uptake (not shown).

[0170] Please note that all compounds A-D increased [³H]-thymidineuptake at least at one concentration value. Increase varied between Ca.1.5 (compound B at 0.03 mM) to Ca. 2.5 (compound D at 0.03 mM and 0.1mM). Thus, all four compounds showed high potency as protectiveextracellular antioxidants. Furthermore, some also reversed the basalcellular oxidation state which occurs spontaneously in control cells(not shown). Thus compounds A-D were proven useful as extracellularantioxidants.

EXAMPLE 8 In vitro Intracellular Antioxidation by Compounds A-D

[0171] One of the main characteristics of the oxidative effects in PC12cells are mimicked by 6-hydroxy-dopamine which is a falseneurotransmitter taken up by the cells. Therefore, 6-hydroxy-dopaminewas used as another oxidative agent and tested the protectiveantioxidant efficiencies of compounds A-D within the cells.

[0172] With reference now to FIG. 2. To this end, PC12 cells weresubjected to high concentration (0.5 mM) of 6-hydroxy-dopamine (6-HO-DA)for 24 hour, in the presence of 0.3 mM or 0.8 mM of compounds A-D or 1mM of reduced glutathione (GSH) a natural antioxidant, as shown in thefront row of FIG. 2. A similar set of cells was treated with the sameconcentrations of compounds A-D and of reduced glutathione, yet without6-hydroxy-dopamine, as shown in the back row of FIG. 2. Due to the highlipophylicity of the antioxidants used, they were first dissolved indimethyl sulfoxide (DMSO), then in water and were applied to the cellsin a final concentration of 3% DMSO. [³H]-thymidine was added to thecells (1 μCi/100,000 cells) six hours prior to the end of the 24 hourperiod.

[0173] The results presented in FIG. 2 are the mean of triplicate wellstaken from three independent cell batches, wherein control representun-treated cells and is defined as 100% [³H]-thymidine uptake. Theeffect of DMSO on the cells was tested separately as shown.

[0174] Please note that all compounds A-D increased [³H]-thymidineuptake of 6-hydroxy-dopamine treated cells, at least at oneconcentration value. Increase varied between Ca. 1.5 (compound C at 0.3mM) to Ca. 3.5 (compound D at 0.3 mM and 0.8 mM). Thus, all fourcompounds showed high potency as protective intracellular antioxidants.Furthermore, some also reversed the basal cellular oxidation state whichoccurs spontaneously in cells not treated with 6-hydroxy-dopamine (FIG.1, back row). Thus compounds A-D were proven useful as intracellularantioxidants.

EXAMPLE 9 In vivo Antioxidation by Compounds A-D

[0175] To demonstrate that indeed compounds A-D cross the blood brainbarrier and affect oxidation state of brain cells, animals were injectedwith compound A and the endogenous reduced glutathione (GSH) amounts inthe serum, in the corpus striatum (at the central nervous system) and/orin the whole brain were determined to evaluate compound A'santioxidation activity within the brain, as was determined by the ratiobetween endogenous brain (corpus striatum) GSH and endogenous serum GSH.

[0176] With reference now to FIG. 3. To this end, three groups of twomonths old Balb/c mice containing two animals in each group wereinjected intraperitonealy (IP) with 100-300 mg/kg body weight ofcompound A. Blood samples were drawn from the tail three hours postinjection and then the animals were sacrificed and either the corpusstriatum or the whole brain were removed and analyzed for GSH levels.

[0177] GSH levels were determined using the experimental procedures asdescribed hereinbelow and/or the GSH-400 kit (Oxis International, Inc.).

[0178] Preparation of brain homogenates. Animals were rapidly killed andexsanguinated to remove excess blood from the brain. The brain of eachanimal was rinsed in a beaker containing water, lightly blotted to dryand were weighted. The striatums were transferred into ahand-homogenizer tube and each was homogenized using a constant number(e.g., 20) of up and down strokes of the hand-homogenizer pestle. Eachof the homogenates was poured into a centrifuge tube and centrifuged for10,000×g for 5 min. The supernatant was used for GSH determination asfollows.

[0179] GSH Assay. For each measurement, 200 μl of sample were incubatedwith 20 μl DTNB [5,5′ dithio bis(2-nitrobenzoic acid)] for 1 hour in 37°C. Final absorbance was measured at 400 nm. Similar results wereobtained using the GSH-400 kit.

[0180] The results shown in FIG. 3 are presented as the OD ratio ofstriatum/serum endogenous GSH levels. Two control mice were injectedwith dimethylsulfoxide (DMSO), since DMSO was used as vehicle for theinjection of compound A. Exogenous GSH was also administered and used asa control for an antioxidant known not to cross the blood brain barrier.

[0181] These results demonstrate that compound A injected IP crosses theblood brain barrier and upon entry to cells at the striatum, increasesthe level of endogenous GSH, demonstrating its potential protectionagainst oxidative stress.

EXAMPLE 10 In vivo Antioxidation by Compound J

[0182] Detection of compound j (referred to in this Example also as CEA)was established using high performance liquid chromatography (HPLC). Tothis end, CEA was treated with a fluorescent thiolyte reagent(monobromobimane reagent) and analyzed on an HPLC column.

[0183] Protection from oxidative stress in vitro: Neuroblastoma SHSY5Y(NB) cells were maintained in Dulbeco's Modified Eagle's Medium (DMEM),supplemented with 8% FCS and 8% horse serum, penicillin (25 μg/ml),streptomycin (25 μg/ml), 2 mM L-glutamine and 400 μg/ml G418(Gibco/BRL). For protection experiments cells were subcultured (in 2%serum) to poly-L-lysine-coated 96-well microtiter plates (Nunc), 100 μlof 5×10⁵ cells/ml, CEA was applied to the cells in each well and 4 hourslater DA, L-dopa (levodopa), 6-OHDA and MPP+ were added for 24 hours.

[0184] Survival was assayed by adding neutral red (0.34%, Sigma) tocells in DCCM-1 medium (0.1 ml/well, Bet-Haemek) and incubation for 2hour at 37° C. The cells were then washed with cold PBS containing 10 mMMgCl₂ and the dye was dissolved in 50% ethanol in 50% Somerson buffer(70 mM sodium citrate, 30 mM citric acid, 0.1 N HCl). ELISA reader (590nm) was used to measure the remaining color intensity.

[0185] Crossing the blood-brain-barrier: In vivo experiments werecarried out on C57BL/6J mice (15 grams) injected IP with compound J.After incubation (15 min, 60 min or 4 hours) the mice were anesthetizedwith ether and blood samples were drawn. Then mice underwent perfusionwith 50 ml of saline, injected into the right ventricle. The levels ofcompound I in the brain and in the plasma were detected by selectivefluorescent labeling using high performance liquid chromatography(HPLC).

[0186] The results are shown in FIGS. 4-6.

[0187] Increasing concentrations of compound J were added to NB cells.compound J at 0.1 mM was found to protect the cells against the toxicity(>50%) of DA, L-dopa (levodopa), 6-OHDA (0.1-0.25 mM) and MPP⁺ (0.5-2mM). Cell survival was increased up to 95% in 0.3 mM compound J asindicated by neutral red assays (FIG. 4).

[0188] Increasing amounts of compound J were injected IP and 15 minlater, mice were perfused with saline and compound J levels in the brainextracts were determined by HPLC chromatography (FIG. 5b), as comparedto a control, pure, uninjected compound J (FIG. 5a).

[0189] IP injection of increasing concentrations (0.25-4 mg) of compoundJ showed a concentration-dependent increase of compound J in the brain(FIG. 6).

[0190] As shown in FIG. 5b, GSH levels were also increased in parallelto compound J presence showing up to 46% increase over untreatedanimals.

[0191] These data indicate that the newly synthesized thiol-substance,compound J, effectively protects cells grown in tissue culture fromoxidative stress. It crosses the BBB as shown by the combinedfluorescent labeling and HPLC chromatography. Furthermore, it increasesendogenous GSH levels in mice brain after IP injection.

[0192] While the invention has been described with respect to a limitednumber of embodiments, it will be appreciated that many variations,modifications and other applications of the invention may be made.

LIST OF REFERENCES IN ALPHABETICAL ORDER

[0193] Brown, D. R., Schmidt, B. & Kretzschmar, H. A. Role of microgliaand host prion protein in neurotoxicity of a prion protein fragment.Nature 380, 345-347 (1996).

[0194] Bundy, G. L., Ayer, D. E., Banitt, L. S., Belonga, K. L., Mizsak,S. A., Palmer, J. R., Tustin, J. M., Chin, J. E., Hall, E. D., Linseman,K. L., et al. Synthesis of novel 2,4-diaminopyrrolo-[2,3-d]pyrimidineswith antioxidant, neuroprotective, and anti asthma activity. J Med Chem.38, 4161-3 (1995).

[0195] Cafe, C., Torri, C., Betorelli, L., Angeretti, N., Luccan E.,Forloni, G., & Marzatico, F., Oxidative stress after acute and chronicapplication of b-amyloid fragment 25-35 in cortical cultures.Neuroscience Letts. 203, 61-65 (1996).

[0196] Craig. C. Transcend therapeutics takes flight against oxidativestress public. BioWorld Today. Aug. 27, 1996, pp.1-2.

[0197] Fahn S. & Cohen, G. The oxidant stress hypothesis in Parkinson'sdisease: evidence supporting it. Ann Neurol. 32, 804-812 (1992).

[0198] Gash., D. M., Zhang, Z., Ovadia, A., Cass, W. A., Simmermann, A.Y. L., Russell., D., Martin, D., Lapchak, P. A., Collins, F., Hoffer, B.J. & Gerhardt, G. A. Furecovery in parkinsonian monkeys treated withGDNF. Nature, 380 252-255 (1996).

[0199] Jenner, O. Oxidative damage in neurodengenerative disease.Lancet, 344, 796-798 (1994).

[0200] Levy, E. G., Anderson, M. E. and Meister A. On the synthesis andcharacterization of N-formylglutathione and N-acetylglutathione. Anal.Biochem., 214, 135-137 (1993).

[0201] Offen, D., Ziv, I., Srernin, H., Melamed, E. and Hochman, A.Prevention of dopamine-induced cell death by thiol-antioxidants:Possible implications for treatment of Parkinson's disease. Exptt.Neurol., 141, 32-39 (1996).

[0202] Olanow, C. W. A. radical hypothesis for neurodegeneration.Trends. Neurol. Soc. 16, 439-444 (1993).

[0203] Olanow, C. W. Oxidation reactions in Parkinson's disease.Neurology 40, 32-37 (1990).

[0204] Sian, J., Dexter, D. T., Lees, A. J.,. Daniel, S., Jenner, P &Marsden, C. D. Glutathione-related enzymes in brain in Parkinson'sdisease. Ann. Neurol. 36, 356-361 (1994).

[0205] Spencer, J. P., Jenner, A., Aruoma, O. I., Evans, P. J. Kauer,H., Dexter, D. T., Jenner, P., Lees, A. J., Marsden, D. C. & Haliwell,B. Intense oxidative DNA damage promoted by L-DOPA and its metabolites.FEBS Letts. 353, 246-250 (1994).

[0206] The Parkinson Study Group. Effects of tocopherol and deprenyl onthe progression of disability in early Parkinson's disease. N. Eng. J.Med. 328, 176-183 (1993).

[0207] Thomas, T., Thomas, G. M., McLendon, C., Sutton, T. & Mullan, M.β-Amyloid-mediated vasoactivity and vascular endothelial damage. Nature380, 168-171 (1996).

What is claimed is:
 1. A method of reducing oxidative stress in thebrain of an organism having a blood brain barrier, the method comprisingadministering an amide compound to the organism, said compound having:(a) a combination of molecular weight and membrane miscibilityproperties for permitting said compound to cross the blood brain barrierof the organism; (b) a sulfhydril group for exerting antioxidationproperties; and (c) a chemical make-up for permitting said compound orits intracellular derivative to accumulate within the cytoplasm ofcells.
 2. The method of claim 1, wherein said administration issystemic.
 3. The method of claim 1, wherein said organism is a humanbeing.
 4. The method of claim claim 1, wherein the oxidative stress inthe brain is a pathology caused by a neurodegenerative disorder.
 5. Themethod as in claim 4, wherein said neurodegenerative disorder isselected from the group consisting of Parkinson's disease, Alzheimer'sdisease, basal ganglia degenerative diseases, motoneuron diseases,Scrapie, spongyform encephalopathy and Creutzfeldt-Jakob's disease. 6.The method as in claim 4, wherein the amide compound is selected fromthe group consisting of:


7. A method of reducing oxidative stress in a tissue of an organism, themethod comprising administering an amide compound to the organism, theamide compound is selected from the group consisting of:


8. The method of claim 7, wherein said organism is a human beingsuffering from a condition associated with over production of oxidants.9. The method of claim 8, wherein said condition is selected from thegroup consisting of acute respiratory distress syndrome, amyotrophiclateral sclerosis, atheroscierotic cardiovascular disease, multipleorgan dysfunction, Parkinson's disease, Alzheimer's disease, basalganglia degenerative diseases, motoneuron diseases, Scrapie, spongyformencephalopathy and Creutzfeldt-Jakob's disease.